INTRODUCTION:

Lithium is a medicine of choice for the therapy of manic-depressive disease^1-4. Therapeutic investigations^6-8 have already been conducted on the metabolic, renal, cerebral, and thyroid functions⁵ of lithium. It is an alkali metal, which can substitute for monovalent (Na⁺, K⁺) and divalent (Mg²⁺, Ca²⁺) cations in various physiological processes. Many studies have examined the interaction between lithium, sodium and potassium ions⁹. In mammals, lithium shares some of its properties of extra cellular sodium and intracellular potassium. Lithium displaces potassium intracellularly and accumulates at the expense of potassium¹⁰.

Long term treatment of lithium has been found to raise plasma calcium in human, rodent and hamsters¹¹ and could cause a depletion in the concentration of free calcium in the cells by increasing the activity of the membrane calcium pump¹².

Lithium can cause a depletion of copper, iron, zinc, and potassium in the bloodstream, according to parallel findings from our laboratory and others.^6,13-15.

Zinc is a part of many biological functions¹⁶. Several studies have also indicated that calcium exerts its antagonistic effect on zinc absorption. Sodium ions provide a driving force needed for the active transport, which is dependent on Na⁺/K⁺ ATPase¹⁷.

Therefore, trace metals, like zinc, act as an important balance for a number of physiological functions that are affected during deficiency states. Sufficient supplementation has the potential to rectify or avert the biochemical and clinical alterations that ensue, as well as function as a pharmacological agent¹⁸. Nevertheless, there is a scarcity of research that has investigated the specific impact of lithium treatment on the interaction between zinc and different elements present in the thyroid and serum. Therefore this study was carried out to understand how lithium affects the levels of Na, K and Ca in blood, Also whether zinc supplementation is useful for normalising the alteration, if any, under such circumstances.

MATERIAL AND METHODS:

Male Wistar rats (150-195g) were obtained from the Central Animal House, Panjab University, Chandigarh and these were acclimatised within the animal house for a week before starting various treatments. The rules of animal care constituted by the National Institute of Health (NIH publication no. 85-23, revised in 1985) were strictly obeyed. Throughout the research, the rats were provided with unrestricted access to food and water and were confined in hygienic polypropylene cages.

The animals were divided into four groups at random. Six to eight rats comprised each cohort, which received distinct treatments for eight weeks. Group-1 rats were provided with ad libitum tap water and standard pellet feed. Group-II animals were given lithium as lithium carbonate in their diet (1.1g/kg body weight)¹⁹. Zinc treatment was administered to the animals in Group III via ingesting water containing zinc sulphate at a concentration of 227mg/L^20,43. In Group IV, zinc and lithium were administered in the same manner as to the animals in Groups 2 and 3, respectively. The p-values represent the levels of significance that are determined through statistical analysis.

Trace Element Analysis:

The trace element analysis was done in the serum. For serum, the blood was withdrawn from the ocular vein of the rats and then serum was subsequently separated via centrifugation of the clotted blood at 3000rpm for 10 minutes. To estimate lithium levels, 0.2ml serum was mixed with 6.25% TCA (1.8 ml), vortexed and were centrifuged at 2000x g for 15 minutes to get the clear supernatant. This process was performed 2-3 times and the supernatant was used for lithium estimation and the absorption maxima was compared with the reading obtained following aspiration of the standard directly into the flame photometer using lithium filter^20,21.

To estimate sodium, potassium and calcium levels, the serum was diluted with double distilled water, sprayed into the flame photometer and the readings were obtained using specific filters as described above¹⁸.

Levels of zinc in the serum was estimated using Perkin Elmer Atomic Absorption Spectrometer Model 4000²².

Statistical Analysis:

The statistical analysis was conducted by applying analysis of variance (ANOVA) and Newman Keul's test. The means and standard deviations were utilised to present the results.

RESULTS:

The serum lithium concentrations of both the lithium-treated group and the combined lithium and zinc-treated group fell within the therapeutic range of 0.32 to 0.70 mEq/L.

The administration of zinc resulted in a significantly increased concentration of zinc (p<0.05) in comparison to the untreated groups. A statistically significant increase in zinc levels (p<0.01) was observed in the combined lithium and zinc treatment group as compared to the lithium treatment group; despite this, no substantial alteration was detected in comparison to the normal group (Table 1).

Table 1: Effect of zinc on serum zinc concentrations in rats administered lithium

Groups		Zinc (μg/g)
I	Normal Controls	35.31 ± 2.90
II	Lithium treated	27.06 ± 2.08^b
III	Zinc treated	40.29 ± 1.73^a
IV	Lithium+Zinc treated	34.56± 2.13^y

Newman- Keuls (q-values)

II vs I	5.04
III vs I	4.73
IV vs I	0.89
IV vs II	8.50

The values are presented as the means±standard deviations of six to eight animals.

^ap< 0.05,^bp< 0.01 through the Newman-Keuls test, which compares the values of groups II, III, and IV to those of group I.

^yp< 0.01 through the Newman-Keuls test, which compares the values of group IV to that of group II.

There was no notable change observed in the zinc concentrations across any of the treatment groups (Table 2).

Table 2: Effect of zinc on serum sodium concentrations following lithium administration to rats

Groups		Sodium (mEq/L)
I	Normal Controls	162.08 ± 5.27
II	Lithium treated	156.71 ± 4.36
III	Zinc treated	158.37 ± 5.92
IV	Lithium+Zinc treated	162.89± 4.31

Newman- Keuls (q-values)

II vs I	2.87
III vs I	1.98
IV vs I	0.44
IV vs II	2.55

The values are presented as the means±standard deviations of six to eight animals.

The groups that received lithium treatment (p<0.01) and combined lithium and zinc treatment (p<0.05) exhibited a significant decrease in potassium levels compared to the control group that received normal conditions. The potassium levels of the zinc-treated group, however, did not change significantly (Table 3).

Table 3: Effect of zinc on serum potassium concentrations following lithium administration to rats

Groups		Potassium (mEq/L)
I	Normal Controls	5.12 ± 0.67
II	Lithium treated	3.77± 0.95^b
III	Zinc treated	4.81 ± 0.64
VII	Lithium+Zinc treated	4.07± 0.72^a

Newman- Keuls (q-values)

II vs I	5.78
III vs I	1.14
VII vs I	3.88
VII vs II	1.06

The values are presented as the means±standard deviations of six to eight animals.

^ap< 0.05,^bp< 0.01 through the Newman-Keuls test, which compares the values of groups II, III, and IV to those of group I.

A statistically significant increase in calcium levels (p<0.001) was observed across all treatment groups when compared to the control group consisting of normal tissue. In contrast, it was determined that the calcium levels were considerably diminished (p<0.05) in comparison to the group that received lithium treatment (Table 4).

Table 4: Effect of zinc on serum calcium concentrations following lithium administration to rats

Groups		Calcium (mEq/L)
I	Normal Controls	5.03 ± 0.16
II	Lithium treated	6.57 ± 0.42^c
III	Zinc treated	4.03 ± 0.21^c
IV	Lithium+Zinc treated	6.06± 0.39^c,x

Newman- Keuls (q-values)

II vs I	14.12
III vs I	8.47
IV vs I	9.11
IV vs II	3.07

The values are presented as the means±standard deviations of six to eight animals.

^cp< 0.001 through the Newman-Keuls test, which compares the values of groups II, III, and IV to those of group I.

^xp< 0.05 through the Newman-Keuls test, which compares the values of group IV to that of group II.

DISCUSSION:

Trace elements are maintained at extreme concentrations to ensure their structural and functional integrity in all living organisms²³. However, any deficiency in these vital elements within the body can result in adverse nutritional and toxicological effects, as well as disrupt the metabolism of other metals^24,25. Additionally, they contribute to trace element deficiencies and alter susceptibility to metal toxicity²⁶. Furthermore, it is widely acknowledged that elements possessing comparable chemical and physical characteristics frequently engage in biological interactions that hinder one another's functionality Given the aforementioned information, the purpose of the current study was to examine the impact of different interventions on the elemental concentrations in the sera of rats.

The lithium and zinc treatment groups, as well as the lithium-treated group, exhibited serum lithium levels within the therapeutic range of 0.32–0.70mEq/L. It has been reported that lithium may exhibit therapeutic efficacy when administered in serum concentrations ranging from 0.6 to 0.8mEq/L to approximately 1.2 to 1.5mEq/L²⁷. Serum lithium concentrations were previously determined to be within the therapeutic range in rats that received lithium at a dose of 1.1g/kg diet; normal rats exhibited no detectable lithium levels^28,29.

Treatment with lithium resulted in a significant reduction in serum zinc levels. Earlier reports from our laboratory had also shown reduced serum zinc levels following lithium administration³⁰. The association of zinc with metal-binding proteins that regulate zinc's functions has been identified. For example, metallothionein is involved in the heavy metal detoxification process and stabilise membranes^31,32. Thus it is possible that during lithium treatment, the levels of metallothionein could have been altered resulting in the decreased levels of zinc.

A substantial increase in zinc levels was observed in the group that received combined lithium and zinc treatment, as compared to the group that received lithium treatment alone, thus indicating the more zinc availability upon its supplementation¹⁹.

Lithium interacts with cations notably Na, K and Ca³³. Sodium is transported via the serosal side of the kidney cell into the interstitial fluid via a Na+/K+ dependent ATPase pump; ATP supplied the energy necessary for this active process. Lithium administration led to slight decrease in the levels of sodium in serum. As lithium, sodium, and potassium ions share similar physical properties, a portion of the therapeutic effects may result from the interaction of lithium with the fluxes of Na and K ions across the RBC membranes³⁴. An individual zinc treatment did not yield a statistically significant alteration in sodium levels when compared to the control group that received normal zinc.

Potassium levels decreased significantly in the present study after lithium supplementation. The decrease in potassium serum concentrations observed in rats treated with lithium validates a previous investigation conducted in our laboratory³⁰. Yurinskaya³⁴ reported that the observed reduction in potassium concentration in astrocytes of experimental rats following lithium administration can be attributed to the inactivation of Na⁺/K⁺ ATPase which is substantiated from the present study where we have seen decreased ATPase activity following lithium treatment. Consequently, this reduced activity subsequent to lithium therapy offers a potential rationale for the diminished potassium concentrations in the serum. A previous report from our laboratory similarly documented a decrease in potassium levels subsequent to lithium treatment^35,36. When lithium was administered to rats treated with zinc, the levels of potassium were found to be reduced, thereby indicating the dominant role of lithium over zinc.

Calcium is an essential element necessary in several cellular processes³⁷. It functions as an intracellular second messenger in transducing an extensive range of enzymes which include adenylate cyclase, glycogen synthase and calcium ATPase³⁷. The present study indicated that lithium administration led to significant increase in serum levels of calcium. Several other scientists also reported an increase in plasma concentration of calcium a short time after lithium supplementation^38,39. As a result of lithium's competition with calcium at the receptor sites, calcium is released into the systemic circulation, according to these authors. An increase in serum calcium levels was also observed subsequent to lithium treatment in the research conducted at our laboratory³⁶. Furthermore, the current study demonstrates that zinc supplementation can reduce serum calcium levels. This phenomenon may be attributed to the antagonistic effect of zinc on calcium⁴⁰, a modification in cellular membrane permeability, or a shift in the activity of membrane-bound enzymes⁴¹. Additionally, there have been reports suggesting that Zn²⁺ can inhibit the Ca²⁺ effect by calcium ion displacement from its cell binding sites⁴². This mechanism alters the membrane calcium pump and ultimately leads to a decrease in free intracellular calcium.The combined lithium and zinc-treated group exhibited decreased calcium levels in comparison to the lithium-treated group, while they increased in comparison to the typical control group. These results suggest that lithium exerts a more pronounced influence on calcium levels than zinc. Therefore, it can be inferred that zinc supplementation in the drinking water of the animals at a concentration of 227mg/L could restore vital trace metals, including potassium and sodium, under the present experimental conditions.

ACKNOWLEDGEMENT:

The authors did not receive grant from any funding agency.

CONFLICT OF INTEREST:

The authors have no relevant financial or non-financial interests to disclose.

AUTHOR CONTRIBUTIONS:

Every author provided input into the formulation and structure of the inquiry. The procedures of data preparation, analysis, and collection were carried out in the presence of Dr. Rajiv Pathak and Dr. Ashima Pathak. The initial draft of the manuscript was authored by Dr. Rajiv Pathak, and all authors contributed their perspectives to a previous version of the document. All authors served as reviewers and approved the final manuscript.

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Received on 05.12.2023 Revised on 19.04.2024

Accepted on 24.07.2024 Published on 24.12.2024

Available online from December 27, 2024

Research J. Pharmacy and Technology. 2024;17(12):5955-5959.

DOI: 10.52711/0974-360X.2024.00903

II vs I

II vs I

II vs I

II vs I